1,721,035 research outputs found
Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells
No full textSilencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable
Heterologous expression of carnation Italian ringspot virus p36 protein enhances necrotic cell death in response to acetic acid in Saccharomyces cerevisiae
No full textA universal feature of the replication of positive-strand RNA viruses is the association with intracellular membranes. Carnation Italian ringspot virus (CIRV) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. The product encoded by CIRV ORF1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. Here the yeast Saccharomyces cerevisiae was used as a model host to study the effect of CIRV p36 on cell survival and death. It was shown that p36 does not promote cell death, but decreases cell growth rate. In addition, p36 changed the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. The tight association of p36 to membranes was not affected by acetic acid treatment, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria in yeast. This work proved yeast as an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses
Mitochondrial Stress Response and Cell adaptation in Saccharomyces cerevisiae as a model organism
No full textDifferent stressful conditions threatening cell homeostasis trigger numerous molecular responses that can result either in cell adaptation to the newly formed conditions and survival, or can cause cell demise. Cell stress response highly depends on the type and level of the insult, but the adaptive capacity of a cell will ultimately determine its fate.
Besides its well-recognized bioenergetic function, mitochondria play an important role in many cell regulatory and signaling events, in the responses of cells to a variety of physiological and genetic stresses, inter-organelle communication, cell proliferation and cell death
Yeast acetic-acid induced programmed cell death resistance in raffinose is controlled by co-operation of ADR1 and CAT8 with the mitochondrial retrograde regulator RTG2
No full textNutrient availability and the differentiation state determine cell fate in multicellular organisms. The yeast Saccharomyces cerevisiae is a valuable model organism to study how nutrient availability determines cell fate in
different growth conditions. In an attempt to understand how metabolism controls cell death and survival in
actively dividing and proliferating cells, we have shown that exponential growing yeast cells in the presence of
the preferred carbon source glucose (GLU-WT), which inhibits respiration through carbon catabolite repression
(CR) pathway, undergo programmed cell death (PCD) in response to acetic acid (AA) treatment. On the contrary,
yeast cells grown in raffinose (RAF-WT) are resistant to AA-PCD in a manner dependent on both the activation
of mitochondrial retrograde (RTG) pathway, which senses mitochondrial dysfunction, and glucose de-repression
of mitochondrial respiration. To study the relationships between CR and RTG pathway in yeast cell death and
survival in response to AA, we compared WT cells and a set of yeast mutants lacking negative or positive
regulators of either RTG or CR pathway as for their viability after AA treatment. We found that glucose-grown
cells lacking MIG1 and HXK2, which repress transcription of mitochondrial respiratory genes in the presence of
glucose, undergo AA-PCD as GLU-WT cells, as judged by decrease in viability and increase in DNA
fragmentation. On the other hand, differently from RAF-WT cells, raffinose-grown cells lacking transcription
factors ADR1 or CAT8, which are activated by alternative carbon sources, or RTG2, a positive regulator of RTG
pathway, underwent AA-PCD. Cells lacking HAP4, the regulatory subunit of HAP complex active in raffinose,
remain fully resistant to AA-PCD. Interestingly, double knock-out ∆adr1∆rtg2 and ∆cat8∆rtg2 cells were found
nearly as much resistant to AA-PCD as RAF-WT cells. RTG pathway activation was studied in raffinose-grown
WT and knock-out cells after AA treatment by analyzing the prototypical RTG-target gene CIT2 mRNA level.
Results showed that ADR1 and CAT8 are positive regulators of RTG2-dependent transcription and suggest that
RTG and CR pathways co-operate in the control of cell fate through interaction between RTG2 and CAT8 or
ADR1
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Stress-related mitochondrial components and mitochondrial genome as targets of anticancer therapy
No full textIn addition to their role as cell powerhouse mitochondria are key organelles in the processes deciding about cell life or death that are crucial for tumor cell growth and survival, as well as for tumor cell ability to metastasize. Alterations in mitochondrial structure and functions have long been observed in cancer cells, thus targeting mitochondria as an anticancer therapeutic strategy has gained momentum recently. We will review the achievements and perspectives in the elucidation of the molecular basis for developing mitochondrial-targeted compounds as potential anticancer agents with special attention to mitochondrial DNA mutations and mitochondrial dysfunction. Molecules/agents candidate to affect mitochondrial metabolism in cancer cells will be dealt with, with a particular focus on approaches targeting defects in the mitochondrial genome
Characterization of the CYP21 gene 5' flanking region in patients affected by 21-OH deficiency
No full textVariations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Yeast as a tool to study mitochondrial retrograde pathway en route to cell stress response
No full textMitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, such as those encoding for peroxisomal citrate synthase, aconitase, and NAD(+)-specific isocitrate dehydrogenase subunit 1, as well as the phosphorylation status of Rtg1/3p transcriptional factor which controls RTG target gene transcription
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